Am. J. Respir. Cell Mol. Biol., Volume 18, Number 2, February, 1998 270-278

Nucleoside Diphosphate Kinase and Cl-sensitive Protein Phosphorylation in Apical Membranes from Ovine Airway Epithelium

Richmond Muimo, Stephen J. Banner, Lindsay J. Marshall, and Anil Mehta

Department of Child Health, Centre for Research into Human Development, Ninewells Hospital Medical School, Dundee, Scotland, United Kingdom

We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl], and anion species determine the steady-state phosphorylation of apical membrane proteins within human airway epithelium in vitro. We found that a Cl-regulated 37-kD protein (p37) principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from sheep tracheal epithelium also contain a Cl-regulated 37-kD phosphoprotein (p37s) and characterize one of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins with [³²P]GTP in the presence of MgCl₂ showed that two proteins circa 19 and 21 kD (p19s and p21s) were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either [³²P]GTP or [³²P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical membrane proteins in the presence of [³²P]ATP and guanosine diphosphate (GDP) (but not GDPS) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg²⁺, Mn²⁺, and Co²⁺ (but not Zn²⁺ or Ca²⁺). Our data show that ovine trachea is a good model for further characterization of the chloride-dependent cascade in airway epithelium.

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